The 1918-19 influenza virus claimed the lives of more than 20 million people worldwide and the determinants of its extreme virulence are currently under investigation. Influenza A virus harbors a segmented, negative-sense RNA genome contained within an envelope that is decorated with 2 surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). The HA mediates both attachment to the host cell via sialic acid and subsequent fusion between the viral envelope and host endosomal membrane. Proteolytic cleavage of HA is a requirement for virus fusion and thus for multicycle viral replication. 1918 influenza virus replication occurs independently of trypsin in cell culture, although it does not contain the multibasic cleavage site within HA normally associated with this feature, such as that found in the highly pathogenic H5 and H7 influenza viruses. Previous work suggests that 1918 NA can facilitate the cleavage of HA, pointing to a mechanism of HA cleavage distinct from H5 and H7 viruses. Our hypothesis is that the ability of NA to facilitate HA cleavage plays a role in the extreme virulence of 1918 influenza virus. The rationale for these studies is based on preliminary data and analogous studies performed with an additional H1N1 virus, A/WSN/33, which point to a role of NA-mediated HA cleavage in pathogenesis. This proposal aims to determine the mechanism of 1918 NA facilitation of HA cleavage, to identify the specific residues in 1918 NA providing this ability, and ultimately, to explore the contribution of this NA activity to pathogenesis. Our approach is to use a combination of virion and cell-based fusion assays to establish a mechanistic model of this phenomenon and to generate recombinant viruses bearing mutant 1918 NAs to determine their effects on 1918 replication in vitro and pathogenesis in vivo. These studies will provide insight into the early events of 1918 influenza virus infection and its mechanism of pathogenesis. By examining the role of NA-mediated HA cleavage in 1918 influenza pathogenesis, we hope to achieve a heightened understanding of the factors that contribute to influenza virulence, which can be monitored in emerging avian influenza viruses. Knowledge of these genotypic signatures of virulence will allow the development of improved predictive measures, prevention techniques, and treatment strategies.